Bacteria engineered for conversion of ethylene to n-butanol

ABSTRACT

The present disclosure provides recombinant bacteria with elevated production of ethanol and/or n-butanol from ethylene. Methods for the production of the recombinant bacteria, as well as for use thereof for production of ethanol and/or n-butanol are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of U.S. ProvisionalApplication No. 62/009,857, filed Jun. 9, 2014, which is herebyincorporated by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with Government support under Grant No.DE-AR0000429 awarded by the Department of Energy. The Government hascertain rights in this invention.

FIELD

The present disclosure provides recombinant bacteria with elevatedproduction of ethanol and/or n-butanol from ethylene. Methods for theproduction of the recombinant bacteria, as well as for use thereof forproduction of ethanol and/or n-butanol are also provided.

BACKGROUND

In the midst of declining fossil fuels reserves and a great expansion ofnatural gas production, increasing effort is seeking to commercializethe conversion of methane into chemical feedstocks and fuels as analternative to petroleum. Large natural gas reservoirs exist throughoutthe world and, thus, have an enormous potential as a clean fuel andchemical feedstock. Currently, the vast majority of natural gas is usedfor heating purposes. This is due largely to the properties of methaneas a heating fuel as well as the difficulty in economically convertingmethane into larger, higher value chemicals and liquid fuels.

Several methods to convert methane indirectly and directly into higherolefins have been report in literature. However, the two large-scalemethods being used commercially are methanol-to-olefins (MTO) and theFischer-Tropsch synthesis (FT) (Alvarez-Galvan et al., Catalysis Today171, 15-23, 2011). Both these processes first involve the generation ofsynthesis gas (Syngas), a mixture of H₂ and CO (Alvarez-Galvan, supra,2011). Syngas production is energy intense, requiring high temperatures(>600° C.), making it a costly process that typically represents 60% oftotal capital costs (Aasberg-Petersen et al., available at:www.topsoe.com/business_areas/methanol/˜/media/PDF%20files/Methanol/Topsoe_large_scale_methanol_prod_paper.ashx, 2008).Thus, direct routes for conversion of methane to olefins and otherchemicals are conceptually preferable and many methods have beenreported (Alvarez-Galvan et al., supra, 2011). However, these reportedmethods suffer from low yield and low product specificity rendering themuneconomical thus far. Therefore, a need exists for a novel, more costeffective route to produce chemical feedstocks and fuels from methane.

The challenges in the overall conversion of methane to chemicals, eitherdirectly or indirectly, largely stem from the activation energy requiredto convert methane into large molecules. This is a result of stabilityand symmetry of methane. Current solutions to activate methane involvethe use of inorganic catalysts such as palladium, which serve to reducethe energy required for methane activation (Aasberg-Petersen et al.,supra, 2008). The use of high temperatures and pressures are alsoneeded. These processes are extremely energy intense and also lackproduct specificity, requiring additional purification steps to separatethe various products.

To circumvent these pitfalls, described herein are biosynthetic pathwaysfor the conversion of ethylene, which may be converted from methane withestablished methods, to acetyl-CoA. The biological assimilation ofethylene has only been reported in methanotrophs (Bull et al., Nature405, 175-178, 2000; and Treude et al., Appl Environ Microbiol 73,2271-2283, 2007). Due to the difficulties in culturing methanotrophs andvery few genetic modification tools, no large-scale applications ofthese organisms has been demonstrated. Therefore, the present disclosuredescribes the construction of ethylene assimilation pathway inrecombinant bacteria, which already have a plethora of available genetictools and has well-established large-scale applications. Furthermore,several examples of pathways for converting basic metabolites to fuelsand chemicals (e.g., acetyl-CoA to n-butanol) in E. coli have beenextensively reported (Rabinovitch-Deere et al., Chemical reviews 113,4611-32, 2013). Thus, by engineering a high flux ethylene assimilationpathway in recombinant bacteria, better performance may be achieved thanwhat has been demonstrated in methanotrophs.

Lastly, since ethylene is already a high volume chemical feedstock usedin the chemical industry, a high performance ethylene assimilationpathway in recombinant bacteria could enable immediate industrialapplications. Due to the broad uses of ethylene as a chemical feedstock,technological innovations for the conversion of methane to ethylene willbe developed. Therefore, a well-engineered ethylene assimilation pathwayin a user-friendly host such as recombinant bacteria will enable thebiological conversion of ethylene into liquid fuels and other high valuechemicals.

SUMMARY

The present disclosure provides recombinant bacteria with elevatedproduction of ethanol and/or n-butanol from ethylene. Methods for theproduction of the recombinant bacteria, as well as for use thereof forproduction of ethanol and/or n-butanol are also provided.

In particular, the present disclosure provides bacteria comprising arecombinant polynucleotide encoding an ethylene hydratase (EH), whereinexpression of the EH results in an increase in production of ethanol ascompared to a corresponding bacterium (e.g., same genus and species)lacking the recombinant polynucleotide. In some embodiments, the EH isan oleate hydratase. In certain embodiments, the oleate hydratase is aLysinibacillus fusiformis oleate hydratase. In some embodiments, the EHis a 2-haloacrylate hydratase. In certain embodiments, the2-haloacrylate hydratase is a Pseudomonas species 2-haloacrylatehydratase. In some embodiments, the EH is a kievitone hydratase. Incertain embodiments, the kievitone hydratase is a Fusarium solanikievitone hydratase. The present disclosure further provides bacteriacomprising a recombinant polynucleotide encoding an alkene monooxygenase(AMO) and an ethylene oxide reductase (EOR), wherein expression of theAMO and the EOR results in an increase in production of ethanol ascompared to a corresponding bacterium (e.g., same genus and species)lacking the recombinant polynucleotide. In some embodiments, the AMO isa toluene monooxygenase. In certain embodiments, the toluenemonooxygenase is a Pseudomonas mendocina toluene monooxygenase. Incertain embodiments, the toluene monooxygenase is a Burkholderia cepaciatoluene monooxygenase. In some embodiments, the EOR is an NAD⁺ dependentformate dehydrogenase. In some embodiments, the recombinantpolynucleotide comprises a first polynucleotide encoding the alkenemonooxygenase (AMO) and a second polynucleotide encoding the ethyleneoxide reductase (EOR). In some embodiments that may be combined with anyof the preceding embodiments, the bacterium further comprises a furtherrecombinant polynucleotide encoding an alcohol/aldehyde dehydrogenase(AADH), wherein expression of either the EH, or the AMO and the EOR, incombination with the AADH results in an increase in production ofacetyl-CoA as compared to a corresponding bacterium (e.g., same genusand species) lacking the recombinant polynucleotides. In certainembodiments, the AADH is an E. coli AdhE. In some embodiments that maybe combined with any of the preceding embodiments, the bacterium furthercomprises a further recombinant polynucleotide encoding an ethanoldehydrogenase (EDH), wherein expression of either the EH, or the AMO andthe EOR, in combination with the EDH results in an increase inproduction of an acetaldehyde as compared to a corresponding bacterium(e.g., same genus and species) lacking the recombinant polynucleotides.In certain embodiments, the EDH is an E. coli AdhP. In some embodiments,the bacterium further comprises a still further recombinantpolynucleotide encoding an acetoaldehyde dehydrogenase (ALDH), whereinexpression of either the EH, or the AMO and the EOR, in combination withthe EDH and the ALDH results in an increase in production of acetyl-CoAas compared to a corresponding bacterium (e.g., same genus and species)lacking the recombinant polynucleotides. In certain embodiments, theALDH is an E. coli MhpF. In certain embodiments, the ALDH is a Listeriamonocytogenes EdgE. In certain embodiments, the EDH is an E. coli AdhP.The present disclosure further provides bacteria comprising arecombinant polynucleotide encoding an alkene monooxygenase (AMO) and anepoxide hydrolase (EPH), wherein expression of the AMO and the EPHresults in an increase in production of ethylene glycol as compared to acorresponding bacterium (e.g., same genus and species) lacking therecombinant polynucleotide. In some embodiments, the bacterium furthercomprises a further recombinant polynucleotide encoding a glycoaldehydereductase (GR), wherein expression of the AMO, the EPH, and the GRresults in an increase in production of glycoaldehyde as compared to acorresponding bacterium (e.g., same genus and species) lacking therecombinant polynucleotides. In some embodiments, the bacterium furthercomprises a still further recombinant polynucleotide encoding aphosphoketolase (PK), wherein expression of the AMO, the EPH, the GR,and the PK results in an increase in production of acetyl-phosphate ascompared to a corresponding bacterium (e.g., same genus and species)lacking the recombinant polynucleotides. In some embodiments, thebacterium further comprises a yet further recombinant polynucleotideencoding a phosphate acetyltransferase (PA), wherein expression of theAMO, the EPH, the GR, the PK, and the PA results in an increase inproduction of acetyl-CoA as compared to a corresponding bacterium (e.g.,same genus and species) lacking the recombinant polynucleotides. In someembodiments, the bacterium further comprises a yet still furtherrecombinant polynucleotides encoding an acetoacetyl-CoA thiolase (AT), a3-hydroxybutyryl-CoA dehydrogenase (HBD), a crotonase (CRT), atrans-enoyl-CoA reductase (TER), and an alcohol/aldehyde dehydrogenase(AADH), wherein expression of the AT, the HBD, the CRT, the TER, and theAADH results in an increase in production of n-butanol as compared to acorresponding bacterium (e.g., same genus and species) lacking therecombinant polynucleotides. In certain embodiments, the AT is an E.coli AtoB. In certain embodiments, the HBD is a C. acetobutylicum Hbd.In certain embodiments, the CRT is a C. acetobutylicum Crt. In certainembodiments, the TER is a T. denticola Ter. In certain embodiments, theAADH is a Clostridium acetobutylicum AdhE2. In some embodiments that maybe combined with any of the preceding embodiments, at least one of therecombinant polynucleotides is stably integrated into the genome of thebacterium. In some embodiments that may be combined with any of thepreceding embodiments, the bacterium is E. coli.

In addition, the disclosure provides methods for producing ethanol. Themethods include: a) providing the bacteria as described in the precedingparagraph; and b) culturing the bacteria of (a) in culture mediumcomprising a substrate under conditions suitable for the conversion ofthe substrate to ethanol, wherein expression of either the EH, or theAMO and the EOR, results in an increase in production of ethanol ascompared to a corresponding bacterium (e.g., same genus and species)lacking the recombinant polynucleotide(s), when cultured under the sameconditions. In some embodiments, the methods further includesubstantially purifying the ethanol. In addition, the disclosureprovides methods for producing n-butanol. The methods include: a)providing the bacteria as described in the preceding paragraph; and b)culturing the bacterium of (a) in culture medium comprising a substrateunder conditions suitable for the conversion of the substrate ton-butanol, wherein expression of the enzymes encoded by the recombinantpolynucleotides results in an increase in production of n-butanol ascompared to a corresponding bacterium (e.g., same genus and species)lacking the recombinant polynucleotides, when cultured under the sameconditions. In some embodiments, the methods further includesubstantially purifying the n-butanol. In some embodiments that may becombined with any of the preceding embodiments, the substrate comprisesethylene. In some embodiments that may be combined with any of thepreceding embodiments, the substrate comprises glucose. In someembodiments that may be combined with any of the preceding embodiments,the substrate comprises ethanol.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates three pathways for the enzymatic production ofacetyl-CoA from ethylene.

FIG. 2 illustrates the three pathways for the enzymatic production ofn-butanol from ethylene.

FIG. 3A-B demonstrates the production of oxirane (also known as ethyleneoxide) from ethene (the IUPAC name for ethylene) using recombinantbacteria. Oxirane (product) and ethene (substrate) levels are shownafter 24 hour incubation. First column: substrate only. Second column:substrate and cells without active enzyme. Third column: no substrate,cells with active enzyme spiked with product. Forth column: substrateand cells with active enzyme, showing substrate depletion and productformation compared to controls.

FIG. 4A-B shows the cell density and n-butanol titer produced by ann-butanol-producing strain. FIG. 4A shows a graph of cell density forn-butanol-producing strain grown in media with no carbon source(triangle with solid line), ethanol (circle with dashed line), orglucose (square dotted and dashed line). “Time” indicates time sinceadding M9 media. FIG. 4B shows n-butanol concentration produced by then-butanol-producing strain after 3 days.

FIG. 5A-B shows the growth rate for E. coli overexpressing MhpF and AdhP(dashed lines) or AdhE (dotted and dashed lines), compared to wild-typeE. coli (NC), when using glucose or ethanol as the carbon source. FIG.5A shows the growth rate of strains in media containing glucose as thecarbon source. FIG. 5B shows the growth rate of strains in mediacontaining ethanol as the carbon source. “Time” indicates time sinceadding M9 media with glucose or ethanol, respectively.

FIG. 6 shows that expression of EdgE and AdhP greatly enhances theproduction of n-butanol from an n-butanol-producing strain grown inmedia containing ethanol. EdgE and AdhP were expressed in the n-butanolpathway as an ethanol assimilating pathway. The strains were incubatedat 37° C. for 24 h to produce n-butanol.

FIG. 7 shows the wild-type (e.g., endogenous) reactions catalyzed byoleate hydratase and 2-chloroacrylate hydratase.

FIG. 8 shows the Gas Chromatography-Flame Ionization Detector (GC-FID)analysis of reactions with ethylene gas and purified oleate hydratase or2-chloroacrylate hydratase.

DETAILED DESCRIPTION

The present disclosure provides recombinant bacteria with elevatedproduction of ethanol and/or n-butanol from ethylene. Methods for theproduction of the recombinant bacteria, as well as for use thereof forproduction of ethanol and/or n-butanol are also provided.

In particular, the present disclosure provides efficient approaches forproducing ethanol and n-butanol using recombinant bacteria.Advantageously, the recombinant bacteria and methods of use describedherein allow the utilization of ethylene to generate higher valuechemicals and liquid fuels. The use of these recombinant bacteria allowsthe generation of these valuable chemicals in a more cost-effectivemanner than existing methods.

Bacteria Engineered for Ethanol and/or n-Butanol Production

The present disclosure provides recombinant bacteria for use in theproduction of ethanol and/or n-butanol. The bacteria contain arecombinant polynucleotide encoding an ethylene hydratase (EH) or arecombinant polynucleotide encoding an alkene monooxygenase (AMO) and anethylene oxide reductase (EOR). Expression of the EH, or the AMO and theEOR, results in an increase in production of ethanol as compared tocorresponding bacteria lacking the polynucleotide (e.g., same genus andspecies) when cultured under the same conditions. Some bacteria containa further recombinant polynucleotide encoding an alcohol/aldehydedehydrogenase (AADH). Expression of either the EH, or the AMO and theEOR, in combination with the AADH results in an increase in productionof acetyl-CoA as compared to corresponding bacteria lacking therecombinant polynucleotides (e.g., same genus and species) when culturedunder the same conditions. Some bacteria contain a still furtherrecombinant polynucleotide encoding an acetoaldehyde dehydrogenase(ALDH). Expression of either the EH, or the AMO and the EOR, incombination with the EDH and the ALDH results in an increase inproduction of acetyl-CoA as compared to corresponding bacteria lackingthe recombinant polynucleotides (e.g., same genus and species) whencultured under the same conditions. The present disclosure also providesrecombinant bacteria containing a recombinant polynucleotide encoding analkene monooxygenase (AMO) and an epoxide hydrolase (EPH). Expression ofthe AMO and the EPH results in an increase in production of ethyleneglycol as compared to corresponding bacteria lacking the polynucleotide(e.g., same genus and species) when cultured under the same conditions.Some bacteria contain a further recombinant polynucleotide encoding aglycoaldehyde reductase (GR). Expression of the AMO, the EPH, and the GRresults in an increase in production of glycoaldehyde as compared tocorresponding bacteria lacking the polynucleotide (e.g., same genus andspecies) when cultured under the same conditions. Some bacteria containa still further recombinant polynucleotide encoding a phosphoketolase(PK). Expression of the AMO, the EPH, the GR, and the PK results in anincrease in production of acetyl-phosphate as compared to correspondingbacteria lacking the polynucleotide (e.g., same genus and species) whencultured under the same conditions. Some bacteria contain a yet stillfurther recombinant polynucleotide encoding a phosphateacetyltransferase (PA). Expression of the AMO, the EPH, the GR, the PK,and the PA results in an increase in production of acetyl-CoA ascompared to corresponding bacteria lacking the polynucleotide (e.g.,same genus and species) when cultured under the same conditions. In someembodiments, any of the above bacteria containing enzyme(s) whoseexpression results in an increase in production of acetyl-CoA mayoptionally contain recombinant polynucleotides encoding anacetoacetyl-CoA thiolase (AT), a 3-hydroxybutyryl-CoA dehydrogenase(HBD), a crotonase (CRT), a trans-enoyl-CoA reductase (TER), and analcohol/aldehyde dehydrogenase (AADH). Expression of the AT, the HBD,the CRT, the TER, and the AADH, in combination with the enzyme(s) whoseexpression results in an increase in production of acetyl-CoA, resultsin an increase in production of n-butanol as compared to correspondingbacteria lacking the polynucleotide (e.g., same genus and species) whencultured under the same conditions.

As detailed above, described herein are bacteria containing twoenzymatic pathways for increased production of ethanol: the ethylenehydratase (EH) pathway, and a pathway including both an alkenemonooxygenase (AMO) and an ethylene oxide reductase (EOR). Furtherdescribed herein are pathways for production of acetyl-CoA which, whencombined with the expression of an acetoacetyl-CoA thiolase (AT), a3-hydroxybutyryl-CoA dehydrogenase (HBD), a crotonase (CRT), atrans-enoyl-CoA reductase (TER), and an alcohol/aldehyde dehydrogenase(AADH), result in the increased production of n-butanol. These pathwaysare (1) EH and AADH; (2) EH, EDH, and ALDH; (3) AMO, EOR, and AADH; (4)AMO, EOR, EDH, and ALDH; and (5) AMO, EPH, GR, PK, and PA.

It is understood that the present disclosure describes the abovepathways as separate only for the purpose of demonstrating that each issufficient to carry out a desired enzymatic pathway. Any enzyme orcombination of enzymes from one of the above pathways may be combinedwith any or all of the components of any of the enzymes from the otherpathways. Stated another way, the pathways delineated above are notstrictly limited to separate embodiments, but may be combined in anycombination to achieve a desired biochemical pathway or production of adesired chemical or combination of desired chemicals, e.g., ethanoland/or n-butanol.

Enzymes

Table I provides the enzymes that have been inserted in variousrecombinant bacteria of the present disclosure.

TABLE I Inserted Enzymes Inserted Enzyme (gene Enzyme NCBI name ifavailable) Source EcoGene No. No. Oleate hydratase Lysinibacillusfusiformis — ZP_07049769 2-haloacrylate hydratase Pseudomonas sp. YL —BAJ13488 Kievitone hydratase Fusarium solani — AAA87627.1 Toluenemonooxygenase Pseudomonas — M65106.1 (T4MO) mendocina Toluenemonooxygenase Burkholderia cepacia — AF349675 (TOM) Alcohol/aldehyde E.coli EG10031 NP_41575.1 dehydrogenase (AdhE) Ethanol dehydrogenase E.coli EG12622 NP_415995.4 (AdhP) Acetoaldehyde E. coli EG13625NP_414885.1 dehydrogenase (MhpF) Acetoaldehyde Listeria monocytogenes —NP_464704.1 dehydrogenase (EdgE) Acetoacetyl-CoA thiolase E. coliEG11672 NP_416728.1 (AtoB) 3-hydroxybutyryl-CoA Clostridium — AAA95971dehydrogenase (Hbd) acetobutylicum Crotonase (Crt) Clostridium —WP_010965999.1 acetobutylicum Trans-enoyl-CoA Treponema denticola —NP_971211.1 reductase (Ter) Alcohol/aldehyde Clostridium — AF321779_1dehydrogenase (AdhE2) acetobutylicum

Several classification schemes exist to enable one of skill to identifyhomologous genes, or proteins with homologous functions or enzymaticproperties, across various bacterial species. Enzymatic reactions can beclassified according to their Enzyme Commission (EC) number. The ECnumber associated with a given enzyme specifies the classification ofthe type of enzymatic reaction that a given enzyme is capable ofcatalyzing. EC numbers do not specify identities of enzymes, but insteadspecify the identity of the chemical reaction that a given enzymecatalyzes. Similarly, proteins can also be assigned Gene Ontology (GO)terms. GO terms attempt to further define the given role and/or functionof a protein in a living organism by specifying protein function interms of a cellular component, a biological process, and/or a molecularfunction. For example, two enzymes from two different species oforganisms that catalyze the same chemical reaction could be assigned thesame EC classification and GO term annotation, despite that therespective enzymes are endogenous to different organisms. EC and GO termclassifications are helpful to those skilled in the art in identifyingthe molecular function and/or activity of a given protein outside ofknowing its unique identifying classification with regard to theorganism it came from, such as its NCBI (National Council forBiotechnology) identifier. EC and GO term classifications may encompassbroad or very narrow enzymatic activities and functions, and manyproteins are classified under several often overlapping EC and GO terms.The classifications listed in this disclosure are included to describeenzymes and genes that could be utilized in certain embodiments. Theyare provided to help those skilled in the art understand the enzymaticactivity or class of interest and are not meant to limit or restrictchoice of enzymes in the embodiments.

Enzymes for Ethanol Production

Certain aspects of the present disclosure relate to bacteria expressingenzymes enabling the conversion of ethylene to ethanol for theproduction of ethanol, as well as their methods of use. Ethylenehydratase (EH) activity converts ethylene (the term “ethene” may be usedinterchangeably herein) to ethanol. While bacteria containing an EHenzyme have been identified, the reaction catalyzing the conversion ofethylene to ethanol has not been identified in nature. Any enzymecatalyzing the formation of ethanol from ethylene may be termed anethylene hydratase of the present disclosure.

In some embodiments, an enzyme characterized to have hydratase activityagainst a substrate related to ethylene may be used as an EH of thepresent disclosure. In some embodiments, the EH is an alkene hydratase.In some embodiments, the EH is an oleate hydratase. Oleate hydratase mayrefer to any enzyme catalyzing the conversion of oleate and water to(R)-10-hydroxystearate. This enzymatic reaction belongs to theclassification EC 4.2.1.53. Oleate hydratases share the molecularfunction of GO term ID GO:0050151. Any protein characterized with theseEC classifications and/or GO terms may possess catalytic oleatehydratase activity. More descriptions of oleate hydratases may be foundin O'Connell et al., Bioengineered 4, 313-321, 2013; Joo et al.,Biochimie 94, 907-915, 2012; Kim et al., Appl Microbiol Biotechnol 95,929-937, 2012; Bevers et al., J Bacteriol 191, 5010-5012, 2009; andKisic et al., Lipids 6, 541-545, 1971. In some embodiments, the oleatehydratase is a Lysinibacillus fusiformis oleate hydratase (see Table Ifor sequence reference).

In some embodiments, the EH is a 2-haloacrylate hydratase.2-haloacrylate hydratase may refer to any enzyme catalyzing theconversion of 2-chloroacrylate to pyruvate using FADH₂ as a co-factor.More description of 2-haloacrylate hydratases may be found in Mowafy etal., Appl Environ Microbiol 76, 6032-6037, 2010. In some embodiments,the 2-haloacrylate hydratase is a Pseudomonas species 2-haloacrylatehydratase (see Table I for sequence reference). In some embodiments, the2-haloacrylate hydratase is a Pseudomonas species strain YL2-haloacrylate hydratase.

In some embodiments, the EH is a kievitone hydratase. Kievitonehydratase may refer to any enzyme catalyzing the conversion of kievitoneand water to kievitone hydrate. This enzymatic reaction belongs to theclassification EC 4.2.1.95. Kievitone hydratases share the molecularfunction of GO term ID GO:0050015. Any protein characterized with theseEC classifications and/or GO terms may possess catalytic kievitonehydratase activity. More descriptions of kievitone hydratases may befound in Li et al., Mol Plant Microbe Interact 8, 388-397, 1995; Turbeket al., Phytochemistry 29, 2841-2846, 1990; Turbek et al., FEMSMicrobiol Lett 73, 187-190, 1992. In some embodiments, the kievitonehydratase is a Fusarium solani kievitone hydratase (see Table I forsequence reference).

Ethylene may also be converted into ethanol using the enzymaticactivities of alkene monooxygenase (AMO) and ethylene oxide reductase(EOR). Alkene monooxygenase may refer to any enzyme catalyzing theconversion of ethylene to ethylene oxide (the term “oxirane” may be usedinterchangeably herein). In some embodiments, an AMO may catalyze theconversion of propene to 1,2-epoxypropane using NADH and oxygen. Thisenzymatic reaction belongs to the classification EC 1.14.13.69. In someembodiments, an AMO may catalyze the production of oxirane (also knownas ethylene oxide) from ethylene. Alkene monooxygenases share themolecular function of GO term ID GO:0018645. Any protein characterizedwith these EC classifications and/or GO terms may possess catalyticalkene monooxygenase activity. More descriptions of alkenemonooxygenases may be found in Ginkel et al., Appl Microbiol Biotechnol24, 334-337 (1986); Coleman and Spain, J Bacteriol 185, 5536-5545(2003); Mattes et al., Arch Microbiol 183, 95-106 (2005); Perry andSmith, J Biomol Screen 11, 553-556 (2006).

In some embodiments, an enzyme characterized to have alkenemonooxygenase activity against a substrate related to ethylene may beused as an AMO of the present disclosure. In some embodiments, the AMOis a toluene monooxygenase. In some embodiments, the toluenemonooxygenase hydratase is a Pseudomonas mendocina toluene monooxygenase(see Table I for sequence reference). In some embodiments, the toluenemonooxygenase hydratase is a Burkholderia cepacia toluene monooxygenase(see Table I for sequence reference). More descriptions of toluenemonooxygenases may be found in McClay et al., Appl Environ Microbiol 66,1877-1882, 2000. In some embodiments, the AMO is a styrenemonooxygenase.

Ethylene oxide reductase (EOR) may refer to any enzyme catalyzing theconversion of ethylene oxide to ethanol using NADH. No NADH dependentepoxide reductase is known in the art. In some embodiments, the EOR is aNAD⁺ dependent formate dehydrogenase. Without wishing to be bound totheory, it is thought that this enzyme may catalyze the reaction sinceits active site is similar in size to ethylene oxide. NAD⁺ dependentformate dehydrogenase may refer to any enzyme catalyzing the conversionof formate to carbon dioxide using NAD+ as a co-factor. This enzymaticreaction belongs to the classification EC 1.2.1.2. NAD⁺ dependentformate dehydrogenases share the molecular function of GO term IDGO:0008863. Any protein characterized with these EC classificationsand/or GO terms may possess catalytic NAD⁺ dependent formatedehydrogenase activity. More descriptions of NAD⁺ dependent formatedehydrogenases may be found in Ferry, FEMS Microbiol Rev 7, 377-382,1990. In some embodiments, the NAD⁺ dependent formate dehydrogenase is aCandida methylica NAD⁺ dependent formate dehydrogenase (see, e.g., NCBICAA57036.1). In some embodiments, the NAD⁺ dependent formatedehydrogenase is a Burkholderia stabilis NAD⁺ dependent formatedehydrogenase (see, e.g., NCBI ACF35003.1). In some embodiments, theNAD⁺ dependent formate dehydrogenase is a Pseudomonas sp. strain 101NAD⁺ dependent formate dehydrogenase (see, e.g., Uniprot P33160).

Enzymes for n-Butanol Production

Certain aspects of the present disclosure relate to bacteria expressingenzymes enabling the conversion of ethylene to n-butanol for theproduction of n-butanol, as well as their methods of use. In someembodiments, bacteria expressing enzymes enabling the production ofethanol (e.g., EH, or AMO and EOR) may further express enzymes enablingthe conversion of this ethanol to acetyl-CoA.

Ethanol may be converted to acetyl-CoA using the enzymatic activity ofan alcohol/aldehyde dehydrogenase (AADH). Alcohol/aldehyde dehydrogenase(AADH) may refer to any enzyme catalyzing the conversion of an alcoholto an aldehyde or ketone using NAD+ as a co-factor and the conversion ofacetaldehyde to acetyl-CoA using NAD+ and CoA as co-factors. Theseenzymatic reactions belong to the classifications EC 1.1.1.1 and EC1.2.1.10. Alcohol/aldehyde dehydrogenases share the molecular functionof GO term IDs GO:0004022 and GO:0008774. Any protein characterized withthese EC classifications and/or GO terms may possess catalyticalcohol/aldehyde dehydrogenase activity. In some embodiments, thealcohol/aldehyde dehydrogenase is an E. coli AdhE (see Table I forsequence reference). In some embodiments, the AADH catalyzes theconversion of butylaldehyde to n-butanol. In some embodiments, thealcohol/aldehyde dehydrogenase is a Clostridium acetobutylicum AdhE2.

Ethanol may also be converted to acetyl-CoA using the enzymaticactivities of an ethanol dehydrogenase (EDH) and an acetoaldehydedehydrogenase (ALDH). Ethanol dehydrogenase (EDH) may refer to anyenzyme catalyzing the conversion of an alcohol to an aldehyde or ketoneusing NAD+ as a co-factor. This enzymatic reaction belongs to theclassification EC 1.1.1.1. Ethanol dehydrogenases share the molecularfunction of GO term ID GO:0004022. Any protein characterized with theseEC classifications and/or GO terms may possess catalytic ethanoldehydrogenase activity. In some embodiments, the ethanol dehydrogenaseis an E. coli AdhP. Acetoaldehyde dehydrogenase (ALDH) may refer to anyenzyme catalyzing the conversion of acetaldehyde to acetyl-CoA usingNAD+ and CoA as co-factors. This enzymatic reaction belongs to theclassification EC 1.2.1.10. Acetoaldehyde dehydrogenases share themolecular function of GO term ID GO: 0008774. Any protein characterizedwith these EC classifications and/or GO terms may possess catalyticacetoaldehyde dehydrogenase activity. In some embodiments, theacetoaldehyde dehydrogenase is an E. coli MhpF. In some embodiments, theacetoaldehyde dehydrogenase is a Listeria monocytogenes EdgE. In apreferred embodiment using the combination of ethanol dehydrogenase andacetoaldehyde dehydrogenase, the bacteria express an E. coli AdhP and aListeria monocytogenes EdgE.

Ethylene may be converted to acetyl-CoA using the enzymatic activitiesof an alkene monooxygenase (AMO), an epoxide hydrolase (EPH), aglycoaldehyde reductase (GR), a phosphoketolase (PK), and a phosphateacetyltransferase (PA). Epoxide hydrolase may refer to any enzymecatalyzing the conversion of an epoxide to glycol. This enzymaticreaction belongs to the classification EC 3.3.2.10. Epoxide hydrolaseshare the molecular function of GO term ID GO:0004301. Any proteincharacterized with these EC classifications and/or GO terms may possesscatalytic epoxide hydrolase activity. Glycoaldehyde reductase may referto any enzyme catalyzing the conversion of ethylene glycol toglycoaldehyde. The general aldehyde reductase enzymatic reaction belongsto the classification EC 1.1.1.21. Aldehyde reductases share themolecular function of GO term ID GO:0004032. Any protein characterizedwith these EC classifications and/or GO terms that acts on ethyleneglycol may possess catalytic glycoaldehyde reductase activity.Phosphoketolase may refer to any enzyme catalyzing the conversion ofglycoaldehyde to acetyl-phosphate. This enzymatic reaction belongs tothe classification EC 4.1.2.9. Phosphoketolases share the molecularfunction of GO term ID GO:0050193. Any protein characterized with theseEC classifications and/or GO terms may possess catalytic phosphoketolaseactivity. Phosphate acetyltransferase may refer to any enzyme catalyzingthe conversion of acetyl-phosphate to acetyl-CoA. This enzymaticreaction belongs to the classification EC 2.3.1.8. Phosphateacetyltransferases share the molecular function of GO term IDGO:0008959. Any protein characterized with these EC classificationsand/or GO terms may possess catalytic phosphate acetyltransferaseactivity.

Certain aspects of the present disclosure relate to bacteria expressingenzymes enabling the conversion of acetyl-CoA to n-butanol for theproduction of n-butanol, as well as their methods of use. Moredescriptions of the synthetic n-butanol pathway are provided in Atsumiet al., Metab Eng 10, 305-311, 2008 and Shen et al., Appl. Environ.Microbiol. 77, 2905-2915, 2011. In some embodiments, bacteria expressingenzymes enabling the increased production of acetyl-CoA also express yetstill further recombinant polynucleotides encoding an acetoacetyl-CoAthiolase (AT), a 3-hydroxybutyryl-CoA dehydrogenase (HBD), a crotonase(CRT), a trans-enoyl-CoA reductase (TER), and an alcohol/aldehydedehydrogenase (AADH). Acetoacetyl-CoA thiolase (also known asacetoacetyl-CoA thiolase) may refer to any enzyme catalyzing theconversion of acetyl-CoA to acetoacetyl-CoA. This enzymatic reactionbelongs to the classification EC 2.3.1.9. Acetoacetyl-CoA thiolasesshare the molecular function of GO term ID GO:0003985. Any proteincharacterized with these EC classifications and/or GO terms may possesscatalytic acetoacetyl-CoA thiolase activity. In some embodiments, theacetoacetyl-CoA thiolase is an E. coli AtoB. 3-hydroxybutyryl-CoAdehydrogenase may refer to any enzyme catalyzing the conversion ofacetoacetyl-CoA to 3-hydroxybutyryl-CoA. This enzymatic reaction belongsto the classification EC 1.1.1.157. 3-hydroxybutyryl-CoA dehydrogenasesshare the molecular function of GO term ID GO:0008691. Any proteincharacterized with these EC classifications and/or GO terms may possesscatalytic 3-hydroxybutyryl-CoA dehydrogenase. In some embodiments, the3-hydroxybutyryl-CoA dehydrogenase is a C. acetobutylicum Hbd. Crotonase(also known as enoyl-CoA hydratase) may refer to any enzyme catalyzingthe conversion of 3-hydroxybutyryl-CoA to crotonyl-CoA. This enzymaticreaction belongs to the classification EC 4.2.1.17. Crotonases share themolecular function of GO term ID GO:0004300. Any protein characterizedwith these EC classifications and/or GO terms may possess catalyticcrotonase activity. In some embodiments, the crotonase is a C.acetobutylicum Crt. Trans-enoyl-CoA reductase may refer to any enzymecatalyzing the conversion of crotonyl-CoA to butyryl-CoA. This enzymaticreaction belongs to the classification EC 1.3.1.38. Trans-enoyl-CoAreductases share the molecular function of GO term ID GO:0019166. Anyprotein characterized with these EC classifications and/or GO terms maypossess catalytic trans-enoyl-CoA reductase activity. In someembodiments, the trans-enoyl-CoA reductase is a T. denticola Ter. Thefinal step in this pathway for n-butanol production may be catalyzed byan AADH as described above.

Bacterial Cells

The present disclosure provides recombinant bacteria. Any culturablebacteria are suitable for use in the compositions and methods describedherein. The term “bacteria” refers to a domain of prokaryotic organisms.Bacteria include at least 11 distinct groups as follows: (1)Gram-positive (gram+) bacteria, of which there are two majorsubdivisions: (1) high G+C group (Actinomycetes, Mycobacteria,Micrococcus, others) (2) low G+C group (Bacillus, Clostridia,Lactobacillus, Staphylococci, Streptococci, Mycoplasmas); (2)Proteobacteria, e.g., Purple photosynthetic+non-photosyntheticGram-negative bacteria (includes most “common” Gram-negative bacteria);(3) Cyanobacteria, e.g., oxygenic phototrophs; (4) Spirochetes andrelated species; (5) Planctomyces; (6) Bacteroides, Flavobacteria; (7)Chlamydia; (8) Green sulfur bacteria; (9) Green non-sulfur bacteria(also anaerobic phototrophs); (10) Radioresistant micrococci andrelatives; (11) Thermotoga and Thermosipho thermophiles.

“Gram-negative bacteria” include cocci, nonenteric rods, and entericrods. The genera of Gram-negative bacteria include, for example,Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella,Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella,Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter,Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium,Chlamydia, Rickettsia, Treponema, and Fusobacterium.

“Gram positive bacteria” include cocci, nonsporulating rods, andsporulating rods. The genera of gram positive bacteria include, forexample, Actinomyces, Bacillus, Clostridium, Corynebacterium,Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus,Nocardia, Staphylococcus, Streptococcus, and Streptomyces.

Although E. coli was utilized in exemplary embodiments, the presentdisclosure is not limited to this genus and species of bacteria. It isunderstood that many bacterial species can be modified to produceethanol and/or n-butanol. It is further understood that variousmicroorganisms can act as “sources” for genetic material encoding targetenzymes (e.g., ethylene hydratases, alkene monooxygenases, ethyleneoxide reductases, alcohol/aldehyde dehydrogenases, ethanoldehydrogenases, acetoaldehyde dehydrogenases, epoxide hydrolases,glycoaldehyde reductases, phosphoketolases, acetyltransferases,acetoacetyl-CoA thiolases, 3-hydroxybutyryl-CoA dehydrogenases,crotonases, and trans-enoyl-CoA reductases) suitable for use inrecombinant bacteria provided herein.

Exemplary embodiments use E. coli as the host bacterial species becauseof the extensive expertise and reagents available for this common modelbacterium. However, protocols and reagents for recombinantly expressingproteins in a wide variety of bacterial host species are known in theart (Current Protocols in Microbiology. Hoboken: Wiley, 2013; Neidhardt,F. C. Escherichia coli and Salmonella: Cellular and Molecular Biology.2^(nd) ed. Washington, D.C.: ASM Press, 1996). Furthermore, because ofthe physiological similarities between bacterial species, existingprotocols and reagents for recombinantly expressing a polypeptide in onespecies (e.g., E. coli) may readily function without undueexperimentation in another species.

Methods of Producing and Purifying Ethanol and n-Butanol

The present disclosure provides methods of producing ethanol orn-butanol. These methods involve providing recombinant bacteria with oneor more recombinant polynucleotides and culturing the recombinantbacteria in a culture medium with a substrate under conditions thatenable the bacteria to convert the substrate into ethanol or n-butanol,thereby producing ethanol or n-butanol in higher amounts or at a higherrate than bacteria of the same species lacking the recombinantpolynucleotides. The methods of the present disclosure may be used toproduce ethanol and n-butanol separately or in combination.

In some embodiments, the methods for producing methods of producingethanol or n-butanol include a culture medium for culturing therecombinant bacteria. “Culture medium” as used herein refers to anycomposition or broth that supports the growth of the bacteria of thepresent disclosure. Suitable culture media may be liquid or solid andcontain any nutrients, salts, buffers, elements, and other compoundsthat support the growth and viability of cells. Common nutrients of aculture medium may include sources of nitrogen, carbon, amino acids,carbohydrates, trace elements, vitamins, and minerals. These nutrientsmay be added as individual components (as in a defined culture medium)or as constituents of a complex extract (for example, yeast extract). Aculture medium may be nutrient-rich to support rapid growth or minimalto support slower growth. A culture medium may also contain any agentused to inhibit the growth of or kill contaminating organisms (e.g., anantibiotic). A culture medium may also contain any compound used tocontrol the activity of an inducible promoter or enzyme (as one example,IPTG may be included to induce expression of any polynucleotidescontrolled by a lac operon or functionally similar promoter). Manyexamples of suitable culture media are well known in the art and includewithout limitation M9 medium, Lysogeny Broth (LB), Terrific Broth (TB),and YT broth.

In some embodiments, recombinant bacteria are cultured. Culturingbacteria refers to providing the bacteria with a suitable nutrientsource (such as a culture medium of the present disclosure) underconditions that allow for bacterial growth. These conditions may includepH, temperature, gas levels (e.g., oxygen and carbon dioxide), pressure,light, and cell density. Suitable ranges for each of these parametersmay differ depending upon the particular bacteria, desired metabolicstate of the bacteria, or the activity of any enzymes expressed by thebacteria. Culturing conditions and methods suitable for a wide range ofbacterial species are well known in the art.

In some embodiments, the culture medium contains a substrate that isconverted by the recombinant bacteria to ethanol or n-butanol. Suitablesubstrates may include any carbon source used by bacteria to produceacetyl-CoA, isobutyryl-CoA, pyruvate, an aldehyde, an ester, or analcohol. In some embodiments, the substrate comprises ethylene. Ethylenemay be added to a suitable nutrient source (such as a culture medium ofthe present disclosure) in any state. In some embodiments, ethylene isadded as a percentage of the gases present in the atmosphere in whichthe bacteria are cultured at a concentration sufficient for n-butanolproduction (for example, without limitation, at 1.5%).

In some embodiments, the substrate comprises glucose, e.g., thesubstrate may be a reduced carbon source that is metabolized by thebacteria via glycolysis into pyruvate or acetyl-CoA (e.g., glucose,glycerol, sugars, starches, and lignocellulosics, including glucosederived from cellulose and C₅ sugars derived from hemicellulose, such asxylose). In some embodiments, the substrate may be ethanol, used in thebacterial production of n-butanol. In some embodiments, the substratemay be an amino acid (e.g., valine or isoleucine) or a compound involvedin an amino acid biosynthesis pathway. A substrate may be a constituentof the culture medium, or it may be exogenously supplemented to theculture medium. A substrate may be continuously present in the culturemedium, or it may be supplemented during bacterial growth. A substratemay be present in any desired amount in the culture medium, dependingupon the metabolic activity and/or output of the bacteria or theirtolerance of the substrate.

In some embodiments, the bacteria are used to produce ethanol. In someembodiments, the bacteria contain polynucleotides encoding an alkenemonooxygenase (AMO) and an ethylene oxide reductase (EOR). The genesencoding the AMO and the EOR components may be part of the samepolynucleotide, or separate polynucleotides. They may be regulated byshared or distinct regulatory elements, such as promoters. In someembodiments, the AMO and EOR genes may be controlled by an induciblepromoter (e.g., the lac operon or a functionally similar element). Insome embodiments, bacteria may be grown in the absence of AMO and EORexpression, then induced to express the AMO and EOR at the same time asuitable substrate for ethanol production is added to the culturemedium. Alternatively, the AMO and EOR may be expressed constitutively.Any other combination of enzymes described herein may be expressed fromthe same polynucleotide, or separate polynucleotides, and regulated inan inducible or constitutive manner.

In some embodiments, the methods of the present disclosure may include astep of substantially purifying the ethanol or n-butanol produced by therecombinant bacteria. In some embodiments, ethanol or n-butanol isevaporated using any gas stripping method known in the art, for exampleby using a Graham condenser. Suitable gas stripping methods need notinclude a heating step. In other embodiments, ethanol or n-butanol maybe distilled from the culture medium. Ethanol or n-butanol may also beextracted from the culture medium using a solvent and distilled from theextract. In some embodiments, ethanol or n-butanol is purified by anyliquid-liquid extraction technique known in the art. In someembodiments, ethanol or n-butanol is purified by any pervaporationtechnique known in the art. Various methods for purifying ethanol orn-butanol from a microbial culture are known in the art (see, e.g., Shenet al., Appl Environ Microbiol, 77, 2905-2915, 2011).

Ethanol or n-butanol may be purified at any step in the culturingprocess. Many methods for product generation and purification frombacterial cultures are known in the art (see, e.g., Villadsen et al.,Bioreaction Engineering Principles. 3^(rd) ed. Springer; 2011). Ethanolor n-butanol purification may be performed continuously duringculturing, as in a continuous culture method, or it may be performedseparately from or after culturing, as in a batch or fed-batch culturemethod.

Supplemental Information

The practice of the present disclosure will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, Molecular Cloning: ALaboratory Manual, second edition (Sambrook et al., 1989);Oligonucleotide Synthesis (Gait, ed., 1984); Animal Cell Culture(Freshney, ed., 1987); Handbook of Experimental Immunology (Weir &Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (Miller &Calos, eds., 1987); Current Protocols in Molecular Biology (Ausubel etal., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al.,eds., 1994); Current Protocols in Immunology (Coligan et al., eds.,1991); The Immunoassay Handbook (Wild ed., Stockton Press NY, 1994);Bioconjugate Techniques (Hermanson, ed., Academic Press, 1996); andMethods of Immunological Analysis (Masseyeff, Albert, and Staines, eds.,Weinheim: VCH Verlags gesellschaft mbH, 1993).

The present disclosure identifies specific polynucleotides/genes usefulin the methods, compositions and organisms of the disclosure. However,it should be recognized that absolute identity to such genes is notnecessary, as substantially similar polynucleotides/genes that performsubstantially similar functions can also be used in the compositions andmethods of the present disclosure. For example, changes in a particulargene or polynucleotide containing a sequence encoding a polypeptide orenzyme can be made and screened for expression and/or activity.Typically such changes include conservative and/or silent mutations.

Due to the inherent degeneracy of the genetic code, polynucleotideswhich encode substantially the same or functionally equivalentpolypeptides can also be used to clone and express the same polypeptides(e.g., enzymes). As will be understood by those of skill in the art, itcan be advantageous to modify a coding sequence to enhance itsexpression in a particular host. The genetic code is redundant with 64possible codons, but most organisms typically use a subset of thesecodons. The codons that are utilized most often in a species are calledoptimal codons, and those not utilized very often are classified as rareor low-usage codons. Codons can be substituted to reflect the preferredcodon usage of E. coli, a process sometimes called “codon optimization”(see, e.g., Murray et al., Nucl Acids Res, 17:477-508, 1989).

Those of skill in the art will recognize that, due to the degeneratenature of the genetic code, a variety of DNA compounds differing intheir nucleotide sequences can be used to encode a given enzyme of thedisclosure. The native DNA sequence encoding the biosynthetic enzymesdescribed above are referenced herein merely to illustrate an embodimentof the disclosure, and the disclosure includes DNA compounds of anysequence that encode the amino acid sequences of the polypeptides andproteins of the enzymes utilized in the methods of the disclosure. Insimilar fashion, a polypeptide can typically tolerate one or more aminoacid substitutions, deletions, and insertions in its amino acid sequencewithout loss or significant loss of a desired activity. The disclosureincludes such polypeptides with different amino acid sequences than thespecific proteins described herein so long as they modified or variantpolypeptides have the enzymatic anabolic or catabolic activity of thereference polypeptide. Furthermore, the amino acid sequences encoded bythe DNA sequences shown herein merely illustrate embodiments of thedisclosure.

In addition, homologs of enzymes useful for generating metabolites areencompassed by the microorganisms and methods provided herein. The term“homologs” used with respect to an original enzyme or gene of a firstfamily or species refers to distinct enzymes or genes of a second familyor species which are determined by functional, structural or genomicanalyses to be an enzyme or gene of the second family or species whichcorresponds to the original enzyme or gene of the first family orspecies. Most often, homologs will have functional, structural orgenomic similarities. Techniques are known by which homologs of anenzyme or gene can readily be cloned using genetic probes and PCR.Homologs can be identified by reference to various databases andidentity of cloned sequences as homolog can be confirmed usingfunctional assays and/or by genomic mapping of the genes.

A protein has “homology” or is “homologous” to a second protein if thenucleic acid sequence that encodes the protein has a similar sequence tothe nucleic acid sequence that encodes the second protein.Alternatively, a protein has homology to a second protein if the twoproteins have “similar” amino acid sequences. Thus, the term “homologousproteins” is defined to mean that the two proteins have similar aminoacid sequences.

As used herein, two proteins (or a region of the proteins) aresubstantially homologous when the amino acid sequences have at leastabout 50% 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity. To determine the percent identity of twoamino acid sequences, or of two nucleic acid sequences, the sequencesare aligned for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second amino acid or nucleicacid sequence for optimal alignment and non-homologous sequences can bedisregarded for comparison purposes). In one embodiment, the length of areference sequence aligned for comparison purposes is at least 50%,typically at least 75%, and even more typically at least 80%, 85%, 90%,95% or 100% of the length of the reference sequence. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”).

The percent identity between the two sequences is a function of thenumber of identical positions shared by the sequences, taking intoaccount the number of gaps, and the length of each gap, which need to beintroduced for optimal alignment of the two sequences. For sequencecomparison, typically one sequence acts as a reference sequence, towhich test sequences are compared. When using a sequence comparisonalgorithm, test and reference sequences are entered into a computer,subsequence coordinates are designated, if necessary, and sequencealgorithm program parameters are designated. Default program parameterscan be used, or alternative parameters can be designated. The sequencecomparison algorithm then calculates the percent sequence identities forthe test sequences relative to the reference sequence, based on theprogram parameters. When comparing two sequences for identity, it is notnecessary that the sequences be contiguous, but any gap would carry withit a penalty that would reduce the overall percent identity. For blastn,the default parameters are Gap opening penalty=5 and Gap extensionpenalty=2. For blastp, the default parameters are Gap opening penalty=11and Gap extension penalty=1.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted using known algorithms (e.g., by thelocal homology algorithm of Smith and Waterman, Adv Appl Math, 2:482,1981; by the homology alignment algorithm of Needleman and Wunsch, J MolBiol, 48:443, 1970; by the search for similarity method of Pearson andLipman, Proc Natl Acad Sci USA, 85:2444, 1988; by computerizedimplementations of these algorithms FASTDB (Intelligenetics), BLAST(National Center for Biomedical Information), GAP, BESTFIT, FASTA, andTFASTA in the Wisconsin Genetics Software Package (Genetics ComputerGroup, Madison, Wis.), or by manual alignment and visual inspection.

A preferred example of an algorithm that is suitable for determiningpercent sequence identity and sequence similarity is the FASTA algorithm(Pearson and Lipman, Proc Natl Acad Sci USA, 85:2444, 1988; and Pearson,Methods Enzymol, 266:227-258, 1996). Preferred parameters used in aFASTA alignment of DNA sequences to calculate percent identity areoptimized, BL50 Matrix 15:−5, k-tuple=2; joining penalty=40,optimization=28; gap penalty-12, gap length penalty=−2; and width=16.

Another preferred example of algorithms suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms (Altschul et al., Nuc Acids Res, 25:3389-3402, 1977; andAltschul et al., J Mol Biol, 215:403-410, 1990, respectively). BLAST andBLAST 2.0 are used, with the parameters described herein, to determinepercent sequence identity for the nucleic acids and proteins of thedisclosure. Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information website. Thisalgorithm involves first identifying high scoring sequence pairs (HSPs)by identifying short words of length W in the query sequence, whicheither match or satisfy some positive-valued threshold score T whenaligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold. These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a word length (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a word lengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix(Henikoff and Henikoff, Proc Natl Acad Sci USA, 89:10915, 1989)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul, ProcNatl Acad Sci USA, 90:5873-5787, 1993). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

Another example of a useful algorithm is PILEUP. PILEUP creates amultiple sequence alignment from a group of related sequences usingprogressive, pairwise alignments to show relationship and percentsequence identity. It also plots a tree or dendogram showing theclustering relationships used to create the alignment. PILEUP uses asimplification of the progressive alignment method (Feng and Doolittle,J Mol Evol, 35:351-360, 1987), employing a method similar to a publishedmethod (Higgins and Sharp, CABIOS 5:151-153, 1989). The program canalign up to 300 sequences, each of a maximum length of 5,000 nucleotidesor amino acids. The multiple alignment procedure begins with thepairwise alignment of the two most similar sequences, producing acluster of two aligned sequences. This cluster is then aligned to thenext most related sequence or cluster of aligned sequences. Two clustersof sequences are aligned by a simple extension of the pairwise alignmentof two individual sequences. The final alignment is achieved by a seriesof progressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. Using PILEUP, a reference sequence is compared to other testsequences to determine the percent sequence identity relationship usingthe following parameters: default gap weight (3.00), default gap lengthweight (0.10), and weighted end gaps. PILEUP can be obtained from theGCG sequence analysis software package, e.g., version 7.0 (Devereaux etal., Nuc Acids Res, 12:387-395, 1984).

Another preferred example of an algorithm that is suitable for multipleDNA and amino acid sequence alignments is the CLUSTALW program (Thompsonet al., Nucl Acids. Res, 22:4673-4680, 1994). ClustalW performs multiplepairwise comparisons between groups of sequences and assembles them intoa multiple alignment based on homology. Gap open and Gap extensionpenalties were 10 and 0.05 respectively. For amino acid alignments, theBLOSUM algorithm can be used as a protein weight matrix (Henikoff andHenikoff, Proc Natl Acad Sci USA, 89:10915-10919, 1992).

Polynucleotides of the disclosure further include polynucleotides thatencode conservatively modified variants of the polypeptides of Table I.“Conservatively modified variants” as used herein include individualmutations that result in the substitution of an amino acid with achemically similar amino acid. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and alleles of thedisclosure. The following eight groups contain amino acids that areconservative substitutions for one another: 1. Alanine (A), Glycine (G);2. Aspartic acid (D), Glutamic acid (E); 3. Asparagine (N), Glutamine(Q); 4. Arginine (R), Lysine (K); 5. Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6. Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7. Serine (S), Threonine (T); and 8. Cysteine (C),Methionine (M).

The terms “derived from” or “of” when used in reference to a nucleicacid or protein indicates that its sequence is identical orsubstantially identical to that of an organism of interest. Forinstance, “an oleate hydratase derived from Lysinibacillus fusiformis,”“an oleate hydratase of Lysinibacillus fusiformis,” or “a Lysinibacillusfusiformis oleate hydratase” refers to an oleate hydratase enzyme havinga sequence identical or substantially identical to a native oleatehydratase enzyme of Lysinibacillus fusiformis. The terms “derived from”and “of” when used in reference to a nucleic acid or protein do notindicate that the nucleic acid or protein in question was necessarilydirectly purified, isolated or otherwise obtained from an organism ofinterest. Thus by way of example, an isolated nucleic acid containing anoleate hydratase coding region of Lysinibacillus fusiformis need not beobtained directly from this species, instead the isolated nucleic acidmay be prepared synthetically using methods known to one of skill in theart.

As used herein in the context of introducing a nucleic acid sequenceinto a cell, the term “introduced” refers to any method suitable fortransferring the nucleic acid sequence into the cell. Such methods forintroduction include but are not limited to protoplast fusion,transfection, transformation, conjugation, and transduction. As usedherein, the term “transformed” refers to a cell that has an exogenouspolynucleotide sequence integrated into its genome or as an episomalplasmid that is maintained for at least two generations.

The terms “coding region,” “open reading frame” and “ORF” refers to asequence of codons extending from an initiator codon (ATG) to aterminator codon (TAG, TAA or TGA), which can be translated into apolypeptide.

In some embodiments of the disclosure, the coding sequences of thepolynucleotides are operably linked to a promoter. In some embodiments,the promoter is an inducible promoter. In some embodiments, the promoteris a constitutive promoter. As used herein, “inducible promoter” refersto a promoter that drives expression of a polynucleotide to which it isoperably linked upon cellular perception of a stimulus. Likewise,inducible promoters can terminate expression of a polynucleotide towhich it is operably linked upon removal of a stimulus. An example of aninducible promoter in the present disclosure is theisopropyl-β-D-thiogalactoside (IPTG) inducible promoter, in which thispromoter drives expression of a polynucleotide to which it is operablylinked upon perception of IPTG, an exogenous chemical. Constitutivepromoters are those promoters that are substantially insensitive toregulation by external stimuli and promote expression of a givenpolynucleotide in an essentially constant manner.

As used herein, “recombinant” or “heterologous” or “heterologouspolynucleotide” or “recombinant polynucleotide” refers to apolynucleotide wherein the exact nucleotide sequence of thepolynucleotide is foreign to (i.e., not naturally found in) a givenhost. These terms may also refer to a polynucleotide sequence that maybe naturally found in a given host, but in an unnatural (e.g., greaterthan or less than expected) amount, or additionally if the sequence of apolynucleotide comprises two or more subsequences that are not found inthe same relationship to each other in nature. For example, regardingthe latter, a recombinant polynucleotide could have two or moresequences from unrelated polynucleotides or from homologous nucleotidesarranged to make a new polynucleotide. Specifically, the presentdisclosure describes the introduction of a recombinant vector into amicroorganism, wherein the vector contains a polynucleotide coding for apolypeptide that is not normally found in the microorganism or containsa foreign polynucleotide coding for a substantially homologouspolypeptide that is normally found in the host organism. With referenceto the host cell's genome, then, the polynucleotide sequence thatencodes the polypeptide is recombinant or heterologous. “Recombinant”may also be used to refer to an organism that contains one or moreheterologous polynucleotides.

As used herein, the term “vector” refers to a polynucleotide constructdesigned to introduce nucleic acids into one or more cell types. Vectorsinclude cloning vectors, expression vectors, shuttle vectors, plasmids,cassettes and the like. As used herein, the term “plasmid” refers to acircular double-stranded DNA construct used as a cloning and/orexpression vector. Some plasmids take the form of an extrachromosomalself-replicating genetic element (episomal plasmid) when introduced intoa host cell.

In some embodiments, at least one of the recombinant polynucleotides isstably integrated into the genome of the bacterium. Some plasmidsintegrate into a host chromosome (integrative plasmid) when introducedinto a host cell, and are thereby replicated along with the host cellgenome. Moreover, certain vectors are capable of directing theexpression of coding regions to which they are operatively linked. Suchvectors are referred to herein as “expression vectors.” Thus expressionvectors cause cells to express polynucleotides and/or polypeptides otherthan those native to the cells, or in a manner not native to the cells).

Genetic modifications that result in an increase in gene expression orfunction can be referred to as amplification, overproduction,overexpression, activation, enhancement, addition, or up-regulation of agene. More specifically, reference to increasing the action (oractivity) of enzymes or other proteins discussed herein generally refersto any genetic modification of the host cell in question which resultsin increased expression and/or functionality (biological activity) ofthe enzymes or proteins and includes higher activity or action of theproteins (e.g., specific activity or in vivo enzymatic activity),reduced inhibition or degradation of the proteins, and overexpression ofthe proteins. For example, gene copy number can be increased, expressionlevels can be increased by use of a promoter that gives higher levels ofexpression than that of the native promoter, or a gene can be altered bygenetic engineering or classical mutagenesis to increase the biologicalactivity of an enzyme or action of a protein. Combinations of some ofthese modifications are also possible.

Genetic modifications which result in a decrease in gene expression, inthe function of the gene, or in the function of the gene product (i.e.,the protein encoded by the gene) can be referred to as inactivation(complete or partial), deletion, interruption, blockage, silencing, ordown-regulation, or attenuation of expression of a gene. For example, agenetic modification in a gene which results in a decrease in thefunction of the protein encoded by such gene, can be the result of acomplete deletion of the gene (i.e., the gene does not exist, andtherefore the protein does not exist), a mutation in the gene whichresults in incomplete or no translation of the protein (e.g., theprotein is not expressed), or a mutation in the gene which decreases orabolishes the natural function of the protein (e.g., a protein isexpressed which has decreased or no enzymatic activity or action). Theterm “functional deletion” as used herein refers to a geneticmodification of a gene that serves to substantially eliminatetranscription, translation or activity of any resulting gene product.More specifically, reference to decreasing the action of proteinsdiscussed herein generally refers to any genetic modification in thehost cell in question, which results in decreased expression and/orfunctionality (biological activity) of the proteins and includesdecreased activity of the proteins (e.g., decreased enzymatic activity),increased inhibition or degradation of the proteins as well as areduction or elimination of expression of the proteins. Combinations ofsome of these modifications are also possible.

The terms “decrease,” “reduce” and “reduction” as used in reference tobiological function (e.g., enzymatic activity, production of compound,expression of a protein, etc.) refer to a measurable lessening in thefunction by preferably at least 10%, more preferably at least 50%, stillmore preferably at least 75%, and most preferably at least 90%.Depending upon the function, the reduction may be from 10% to 100%. Theterm “substantial reduction” and the like refers to a reduction of atleast 50%, 75%, 90%, 95% or 100%.

The terms “increase,” “elevate” and “elevation” as used in reference tobiological function (e.g., enzymatic activity, production of compound,expression of a protein, etc.) refer to a measurable augmentation in thefunction by preferably at least 10%, more preferably at least 50%, stillmore preferably at least 75%, and most preferably at least 90%.Depending upon the function, the elevation may be from 10% to 100%; orat least 10-fold, 100-fold, or 1000-fold up to 100-fold, 1000-fold or10,000-fold or more. The term “substantial elevation” and the likerefers to an elevation of at least 50%, 75%, 90%, 95% or 100%.

The terms “isolated” and “purified” as used herein refers to a materialthat is removed from at least one component with which it is naturallyassociated (e.g., removed from its original environment). The term“isolated,” when used in reference to a biosythetically-produced ester,refers to an ester that has been removed from the culture medium of thebacteria that produced the ester. As such an isolated ester is free ofextraneous or unwanted compounds (e.g., substrate molecules, bacterialcomponents, etc.).

As used herein, the singular form “a”, “an”, and “the” includes pluralreferences unless indicated otherwise.

The phrase “comprising” as used herein is open-ended, indicating thatsuch embodiments may include additional elements. In contrast, thephrase “consisting of” is closed, indicating that such embodiments donot include additional elements (except for trace impurities). Thephrase “consisting essentially of” is partially closed, indicating thatsuch embodiments may further comprise elements that do not materiallychange the basic characteristics of such embodiments. It is understoodthat aspects and embodiments described herein as “comprising” include“consisting” and/or “consisting essentially of” aspects and embodiments.

Examples

To better facilitate an understanding of embodiments of the disclosure,the following examples are presented. The following examples are merelyillustrative and are not meant to limit any embodiments of the presentdisclosure in any way.

Abbreviations: ethylene hydratase (EH); alkene monooxygenase (AMO);ethylene oxide reductase (EOR); alcohol/aldehyde dehydrogenase (AADH);ethanol dehydrogenase (EDH); acetoaldehyde dehydrogenase (ALDH); epoxidehydrolase (EPH); glycoaldehyde reductase (GR); phosphoketolase (PK);phosphate acetyltransferase (PA); acetoacetyl-CoA thiolase (AT);3-hydroxybutyryl-CoA dehydrogenase (HBD); crotonase (CRT);trans-enoyl-CoA reductase (TER); and alcohol/aldehyde dehydrogenase(AADH).

Introduction

Described herein are methods for engineering ethylene assimilationpathways into the industrial host Escherichia coli to convert ethyleneinto acetyl-CoA. In order to maximize the chances of a successfultransplant into E. coli and to optimize efficiency, three alternativesbased on engineered enzymes have been used to generate acetyl-CoA (FIG.1). This acetyl-CoA may be converted into n-butanol through a knownenzymatic pathway (FIG. 2).

As shown in FIG. 1, for pathway 1, the initial step is to convertethylene into ethanol by ethylene hydratase (EH). For pathways 2 and 3,the initial step in the biological assimilation of ethylene is anepoxidation reaction that produces ethylene oxide by alkenemonooxygenase. Several alkene monooxygenases from bacteria have beenidentified (Ginkel et al., Appl Microbiol Biotechnol 24, 334-337, 1986;Coleman and Spain, J Bacteriol 185, 5536-5545, 2003; Mattes et al., ArchMicrobiol 183, 95-106, 2005; Perry and Smith, J Biomol Screen 11,553-556, 2006). However, very little characterization of the catalyticproperties of this enzyme has been done. These pathways are differentfrom the natural ethylene assimilating pathway in Nocardioides sp.strain JS614, which is the only ethylene assimilating pathway identifiedin nature (Mattes et al., Arch Microbiol 183, 95-106, 2005). For Pathway2, a novel NADH dependent epoxide reductase is used to catalyze theconversion of ethylene oxide to ethanol. The resulting ethanol canreadily be converted into n-butanol via acetyl-CoA. In the thirdpathway, ethylene is converted to acetyl-coA via acetyl-phosphate. Theadvantage of this pathway is that all of the required enzymes functionsexist in nature. Since ethylene assimilation to acetyl-CoA is redoxbalanced, this pathway may be introduced into E. coli without affectingits energy metabolism. From acetyl-CoA, n-butanol can be produced usinga pathway known in the art (Atsumi et al., Metab Eng 10, 305-311, 2008).

Materials and Methods Reagents and Bacterial Strains

Restriction enzymes and antarctic phosphatase were from New EnglandBiolabs (Ipswich, Mass., USA). Rapid DNA ligation kit was from Roche(Mannheim, Germany). KOD DNA polymerase was from EMD Chemicals (SanDiego, Calif., USA). Oligonucleotides were from Integrated DNATechnologies (San Diego, Calif., USA).

TABLE 1 E. coli Strains and Plasmids Strain/Plasmid Description BW25113rrnBT14 ΔlacZWJ16 hsdR514 ΔaraBADAH33 ΔrhaBADLD78 TG1 supE hsdΔ5thiΔ(lac-proAB) F′[traD36 proAB⁺ lacI^(q) lacZΔM15] JCL16 BW25113F′[traD36 proAB⁺ lacI^(q)ZΔM15] JCL299 Same as JCL16 but ΔldhA ΔadhEΔfrdBC Δpta pEL11 PLlacO1::atoB_(Ec)-adhE2_(Ca)-crt_(Ca)-hbd_(Ca) ColE1ori Amp^(r) pIM8 PLlacO1::ter_(Td) Cola ori Kan^(r) pAL812 PLlacO1::adhP p15A ori Kan^(r) pAL811 PLlacO1:: mhpF p15A ori Kan^(r) pAL726PLlacO1:: mhpF-adhP p15A ori Kan^(r) pAL727 PLlacO1:: adhE_(Ec) p15A oriKan^(r) pAL728 PLlacO1:: adhE1_(Ca) p15A ori Kan^(r) pAL729 PLlacO1::adhE2_(Ca) p15A ori Kan^(r) pAL885 PLlacO1:: mhpF-mhpE-adhP p15A oriKan^(r) pAL881 PLlacO1:: edgE_(Lm)-adhP p15A ori Kan^(r) pAL896PLlacO1:: edgE_(Lm)-adhP p15A ori Spec^(r) pBS(Kan)ToMO Plac::touABCDEF_(Ps) ColE1 ori Kan^(r) pAL892 PLlacO1::ter_(Td)-touABCDEF_(Ps) Cola ori Kan^(r) Ec: from Escherichia coli, Ca;from Clostridium acetobutylicum, Lm: from Listeria Monocytogenes, Lf:Lysinibacillus fusiformis.

Cell Culture

For standard culturing purposes, E. coli was grown in 5-20 mL of LBmedia containing any required antibiotics at 37° C. with shaking at 250rpm in a rotary shaker. For production experiments, E. coli was culturedin LB media to an OD₆₀₀ of 0.4, then IPTG (final concentration; 1 mM)was added into the culture to induce enzymes in n-butanol pathway andincubated at 30° C. After 1 h, the culture was spun down, and thesupernatant was eliminated. The same volume of M9 media without carbonsource as the LB media was added and the cells were resuspended.Consequently, glucose or ethanol was added as a solo carbon source intothe culture (the final concentration was 10 g/L each). These cultureswere incubated at 30° C. for n-butanol production. OD₆₀₀ and n-butanolconcentration of the culture were measured at specific time point(s).

Computational Tools for Protein Design

Computational protein design is a rapidly evolving method for findingproteins with functions not existing in Nature. The use of computationaltools allows rapid in silico screening of a vast proteinsequence-structure-function space, allowing one to identify a subset ofprotein sequences likely containing the structure/function of interest.This virtual screening enables one to quickly make large jumps insequence space and introduce novel function and structure into aprotein.

The Rosetta Molecular Modeling Suite has been successfully used for theengineering of many proteins (Schueler-Furman et al., Science 310,638-642, 2005). For example: (1) the design of highly stabilizedvariants of naturally occurring proteins (Dantas et al., J Mol Biol 332,449-460, 2003; Dantas et al., J Mol Biol 366, 1209-1221, 2007; Borgo andHavranek, Proc Natl Acad Sci USA 109, 1494-1499, 2012), (2) the designof a protein fold not observed in nature (Kuhlman et al., Science 302,1364-1368, 2003), (3) the specificity redesign and de novo design ofprotein-protein interactions (Chevalier et al., Mol Cell 10, 895-905,2002); Kortemme et al., Nat Struct Mol Biol 11, 371-379, 2004;Joachimiak et al., J Mol Biol 361, 195-208, 2006; Fleishman et al.,Science 332, 816-821, 2011), (4) the design of homing endonucleases withnovel specificity (Ashworth et al., Nature 441, 656-659, 2006; Thyme etal., Nature 461, 1300-1304, 2009), (5) the design of novel enzymescatalyzing chemical reactions for which natural enzymes are notoptimized (Zanghellini et al., Protein Sci 15, 2785-2794, 2006; Jiang etal., Science 319, 1387-1391, 2008; Rothlisberger et al., Nature 453,190-194, 2008; Siegel et al., Science 329, 309-313, 2010), and (6) thedesign of large self-assembling macromolecular structures (King et al.,Science 336, 1171-1174, 2012). Because the designed protein sequences donot exist in Nature, gene synthesis is central to obtaining the designedprotein, and allows one to optimize for expression in any desired host.

The general strategy of computational enzyme design is to use a forcefield composed of potentials derived empirically (van der Waalsinteractions, orientation dependent hydrogen bonding, Coulombelectrostatics, and implicit solvation) and statistically (Ramachandranangles, side chain torsions, pair potentials, etc.) to evaluate howsubstitutions in the protein affect the protein stability andstabilization of the transition state of the desired reaction. Rosettasearches heuristically through combinations of mutations, evaluating theenergetics for each set of mutations. It returns the set of mutationsdetermined to be most favorable for both folding and transition statestabilization.

Example 1: Conversion of Ethylene to Ethanol Using Ethylene Hydratase

The wasteful expenditure of energy in the activation O₂ for epoxidationof ethylene to ethylene oxide, the branch point for the three originallyproposed pathways for conversion of ethylene into n-butanol, canpotentially be circumvented by the development of an enzyme that ishighly active in the hydration of ethylene directly to ethanol (FIG. 1).Interestingly, very few hydration reactions are carried out on isolateddouble bonds in biology. Nearly all biological hydration reactions occuron activated alkenes that are conjugated to groups that can resonancestabilize carbanions formed in Michael-type addition reactions.

An important point is that the hydration of ethanol is carried out on alarge scale industrially and is responsible for approximately half ofthe total world production of ethanol annually. The industrial processinvolves a high temperature gas-phase acid catalyzed hydration reactionand results in a low single-pass yield. The remaining unreacted ethyleneis isolated and rerun through the reactor. Given the inefficiency of thecurrent industrial ethylene hydration process, there may well be asubstantial market for a high efficiency, low temperature enzymecatalyzed process. Therefore, the methods described herein for theconstruction of pathways 1 and/or 2 may also be used for ethanolproduction.

Work on this pathway was initiated by meticulously searching theliterature for enzymes that catalyze hydration of isolated, unconjugateddouble bonds. A thorough search of the literature led to identificationof two classes of known alkene hydratases. The first class employs FADH(reduced flavin adenine dinucleotide) as a cofactor. This ismechanistically interesting since the hydration reaction involves no netredox change in the substrate. Two enzymes in this first class that havebeen investigated are oleate hydratase and 2-haloacrylate hydratase(O'Connell et al., Bioengineered 4, 313-321, 2013; Joo et al., Biochimie94, 907-915, 2012; Kim et al., Appl Microbiol Biotechnol 95, 929-937,2012; Bevers et al. J Bacteriol 191, 5010-5012, 2009; and Kisic et al.,Lipids 6, 541-545, 1971). The second class of enzymes is involved in thehydration of terminal alkenes of carotenoids and other natural products.This class includes carotenoid hydratases, kievitone hydratase, andphaseollidin hydratase (Li et al., Mol Plant Microbe Interact 8,388-397, 1995; Turbek et al., Phytochemistry 29, 2841-2846, 1990; Turbeket al., FEMS Microbiol Lett 73, 187-190, 1992; Sun et al., Microbiology155, 2775-2783, 2009; Steiger et al., Arch Biochem Biophys 414, 51-58,2003).

To date several target enzymes believed to be suitable templates for anethylene hydratase have been identified. E. coli optimized syntheticgenes for three (kievitone hydratase, oleate hydratase, and2-haloacrylate hydratase) have been prepared, and two of these have beencloned (oleate hydratase and 2-haloacrylate hydratase) into the pET28aexpression vector. These genes have been sequenced to confirm thecorrect sequences, transformed into E. coli, and shown by SDS-PAGE toyield high level expression of the expected enzymes (data not shown).The specific enzymes investigated are the oleate hydratase (ZP_07049769)from Lysinibacillus fusiformis, the 2-haloacrylate hydratase (BAJ13488)from Pseudomonas sp. and the kievitone hydratase (AAA87627.1) fromFusarium solani, since these are all well expressed in E. coli and theiractivities have been well characterized. Future experiments will purifythe oleate and 2-haloacrylate hydratases, assay their activities ontheir natural substrates to ensure they are fully active, and then testfor activity with ethylene and other simple alkenes such as propene andbutane.

Example 2: Conversion of Ethylene to Ethanol Using AMO and EOR

The initial step in the biological assimilation of ethylene for pathways2 and 3 (FIG. 1B) is an epoxidation reaction that produces ethyleneoxide by a monooxygenase. In native ethylene utilization pathways, thefirst enzyme is alkene monooxygenase, AMO (Ginkel et al., Appl MicrobiolBiotechnol 24, 334-337, 1986; Coleman and Spain, J Bacteriol 185,5536-5545, 2003; Mattes et al., Arch Microbiol 183, 95-106, 2005; Perryand Smith, J Biomol Screen 11, 553-556, 2006). While several isozymeshave been identified, and one heterologously expressed and active invivo (Perry and Smith, supra, 2006), none have been demonstrated tofunction in E. coli. Therefore, in addition to pursuing AMO, the use ofthe structurally related toluene monooxygenase (TMO) will also betested, as well as the functionally related but structurally dissimilarstyrene monooxygenase (SMO). Both of these alternative enzymes are wellcharacterized and known to function in E. coli. Details discussing eachof the three proposed routes to achieve ethylene oxidation are discussedbelow.

Previous efforts have demonstrated that one potential reason AMO was notfunctional in E. coli was an abundance of rare codons and non-ideal E.coli ribosome binding site (RBS) motifs (Smith et al., Eur J Biochem260, 446-452, 1999). In fact, some of the component proteins were noteven observed, suggesting that translation was not efficientlyoccurring. However, the recombinant AMO has been functionally producedin S. lividans, showing that if all the AMO components are expressedthey can form a functional complex in a heterologous host (Smith et al.,supra, 1999). Therefore, the AMO operon will be resynthesized such thatboth the gene composition and operon structure will be optimized for E.coli. E. coli optimized genes for each component part will be combinedusing Gibson assembly (Gibson et al., Nat Methods 6, 343-345, 2009) inwhich an idealized RBS, designed using the RBS calculator (Salis et al.,Nat Biotechnol 27, 946-950, 2009), will be introduced before each gene.While there are clearly many possible ways to design and assemble theoperon, this simple design is likely to at least enable production ofeach polypeptide to determine if AMO can be functionally produced at anylevel in E. coli. Upon AMO production, the RBS will be further optimizedby adjusting gene order and direction, RBS strength, as well as promoterand terminator placements (Temme et al., Proc Natl Acad Sci USA 109,7085-7090, 2012).

While the use of the native AMO gene would be ideal, closely relatednon-heme diiron enzymes such as methane monooxygenase have never beenfunctionally produced in E. coli despite decades of effort (TorresPazmino et al., J Biotechnol 146, 9-24, 2010). However, the enzymetoluene monooxygenase is a closely related non-heme diiron-dependentmonooxygenases, sharing the (αβγ)₂ quaternary structure (Small andEnsign, J Biol Chem 272, 24913-24920, 1997), and sharing wellestablished functional production in E. coli (Pikus et al., Biochemistry35, 9106-9119, 1996; Studts et al., Protein Expr Purif 20, 58-65, 2000).Furthermore, toluene monooxygenase has been reported to have rates foralternative substrates, such as 2-butene, on the order of 5μmol/g_(total cell protein)/s (McClay et al., Appl Environ Microbiol 66,1877-1882, 2000).

While a similar rate for ethylene is expected to be observed, this canlikely be improved with enzyme engineering, as TMO has not beennaturally evolved to function on ethylene or butene. If rates onethylene are not greater than 10 μmol/g_(total cell protein)'s, twolines of enzyme engineering will be pursued. First, known crystalstructures of product or substrate bound TMO (Bailey et al.,Biochemistry 51, 1101-1113, 2012) will be used to re-engineer the enzymeactive site using the computational design techniques described above.In parallel, due to the high structural and functional similaritybetween TMO and AMO, the recently developed algorithm JANUS, which hasbeen demonstrated to be capable of interconverting enzyme functions(Addington et al., J Molec Biol, 425, 1378-1389, 2013), will be used.This is an ideal example of utility of this algorithm in which the aimwill be to transfer the reaction specificity of AMO into thestructurally and functionally related enzyme TMO.

An alternative to the diiron group of enzymes is a group offlavin-dependent monooxygenases, including styrene monooxygenase, whoseX-ray structure has been determined (Ukaegbu et al., Biochemistry 49,1678-1688, 2010). This FAD dependent enzyme has activity on a variety ofalkenes including 1-hexene, which is as good as styrene as a substrate(Toda et al., Appl Microbiol Biotechnol 96, 407-418, 2012). The kineticsof the enzyme have been investigated in detail (Kantz and Gassner,Biochemistry 50, 523-532, 2011). A natural fusion with the FAD reductasecomponent exists, simplifying its application here (Tischler et al.,Appl Biochem Biotechnol 167, 931-944, 2012). The active site of thisenzyme will be engineered to improve specificity for ethylene, andachieving high level expression in E. coli.

Two toluene monooxygenases (T4MO from Pseudomonas mendocina: M65106.1,TOM from Burkholderia cepacia: AF349675) were tested for theiractivities on ethylene. Cell cultures were incubated at 37° C. in a 1.5%ethylene atmosphere. Under these conditions, ethylene is converted intooxirane (ethylene oxide). FIG. 3 demonstrates that toluene monooxygenaseactivity has been detected in this system using the enzyme T4MO fromPseudomonas mendocina: M65106.1.

Several other potential alkene monooxygenases, including an alkenemonooxygenase from Xanthobacter strain Py2 and an alkene monooxygenasefrom Nocardia corallina B-276, will also be tested. A P450 fromXanthobacter autotrophius, an autotrophic organism that grows onethylene, has been identified and will be tested for ethylene activity(it has been shown to work on halogenated ethenes).

For these pathways, a novel NADH dependent epoxide reductase, EOR, willbe designed to catalyze the conversion of ethylene oxide to ethanol. Theresulting ethanol will be converted to acetyl-coA by well-characterizedethanol dehydrogenase and acetaldehyde dehydrogenase (FIG. 1). No NADHdependent epoxide reductase is known in nature. The reactionthermodynamics are highly favorable (>10 kcal/mol), and NADH dependentreductases are common. NAD⁺ dependent formate dehydrogenase will beinvestigated (Ferry, FEMS Microbiol Rev 7, 377-382, 1990), since theactive site is similar in size to ethylene oxide, and it is awell-studied enzyme with multiple ligand-bound crystal structuresavailable (Schirwitz et al., Protein Sci 16, 1146-1156, 2007; Shabalinet al., Acta Naturae 1, 89-93, 2009). In addition, it is a proficientenzyme with specific activities reported to be >7000μmol/g_(total cell protein)/s (Lu et al., Appl Microbiol Biotechnol 86,255-262, 2010).

A functional search for NAD- and NADP-dependent enzymes revealed aboutthirty thousand candidates for scaffolds on which to build an enzyme toconvert oxirane into ethanol. Filtering out ferridoxin- andmetal-dependent enzymes brings the list down to 200. A group of thesethat may be good candidates as scaffolds for oxirane reductase werecompiled (Table 2). Candidates were chosen based on several criteria: 1)catalyzes a simple hydride transfer, 2) no DHFR, 3) metal independentenzymes, and 4) less than 500 amino acids in a monomer. Synthetic genesfor these candidates will be tested.

TABLE 2 PDB ID Citation 2AZN X-RAY Structure of2,5-diamino-6-ribosylamino-4(3h)-pyrimidinone 5-phosphate reductase 1NNUCrystal Structure Analysis of Plasmodium falciparumenoyl-acyl-carrier-protein reductase with Triclosan Analog 3ORF CrystalStructure of Dihydropteridine Reductase from Dictyostelium discoideum3RJ5 Structure of alcohol dehydrogenase from Drosophila lebanonesisT114V mutant complexed with NAD+ 1OAA MOUSE SEPIAPTERIN REDUCTASECOMPLEXED WITH NADP AND OXALOACETATE 2AG8 NADP complex ofPyrroline-5-carboxylate reductase from Neisseria meningitidis 1SNYCarbonyl reductase Sniffer of D. melanogaster 2F1K Crystal structure ofSynechocystis arogenate dehydrogenase 2G5C Crystal Structure ofPrephenate Dehydrogenase from Aquifex aeolicus 3JYO Quinatedehydrogenase from Corynebacterium glutamicum in complex with NAD 1LUAStructure of methylene-tetrahydromethanopterin dehydrogenase fromMethylobacterium extorquens AM1 complexed with NADP 1LC3 CrystalStructure of a Biliverdin Reductase Enzyme-Cofactor Complex 1YJQ Crystalstructure of ketopantoate reductase in complex with NADP+ 3ZHB R-iminereductase from Streptomyces kanamyceticus in complex with NADP. 3AJRCrystal structure of L-3-Hydroxynorvaline bound L-Threoninedehydrogenase (Y137F) from Hyperthermophilic Archaeon Thermoplasmavolcanium 1EE9 CRYSTAL STRUCTURE OF THE NAD-DEPENDENT 5,10-METHYLENETETRAHYDROFOLATE DEHYDROGENASE FROM SACCHAROMYCES CEREVISIAECOMPLEXED WITH NAD 1E6U GDP 4-KETO-6-DEOXY-D-MANNOSE EPIMERASE REDUCTASE2DBQ Crystal Structure of Glyoxylate Reductase (PH0597) from Pyrococcushorikoshii OT3, Complexed with NADP (I41) 3KVO Crystal structure of thecatalytic domain of human Hydroxysteroid dehydrogenase like 2 (HSDL2)2I3G Crystal structure of N-Acetyl-gamma-Glutamyl-Phosphate Reductase(Rv1652) from Mycobacterium tuberculosis in complex with NADP+. 2ZB4Crystal structure of human 15-ketoprostaglandin delta-13-reductase incomplex with NADP and 15- keto-PGE2 2V6G STRUCTURE OF PROGESTERONE5BETA-REDUCTASE FROM DIGITALIS LANATA IN COMPLEX WITH NADP 3PZR Crystalsstructure of aspartate beta-Semialdehyde dehydrogenase from VibrioCholerae with NADP and product of S-carbamoyl-L-cysteine 1O2D Crystalstructure of Alcohol dehydrogenase, iron-containing (TM0920) fromThermotoga maritima at 1.30 A resolution 2D2I Crystal Structure ofNADP-Dependent Glyceraldehyde-3-Phosphate Dehydrogenase fromSynechococcus Sp. complexed with Nadp+ 3OET D-Erythronate-4-PhosphateDehydrogenase complexed with NAD 1KOL Crystal structure of formaldehydedehydrogenase 1VLJ Crystal structure of NADH-dependent butanoldehydrogenase A (TM0820) from Thermotoga maritima at 1.78 A resolution1L18 Crystal structure of mannitol dehydrogenase in complex with NAD

Example 3: Conversion of Ethylene to Acetyl-CoA Through Ethylene Glycol

In the third pathway, ethylene is converted to acetyl-coA viaacetyl-phosphate (FIG. 1). The advantages of this pathway are that nocofactor biosynthetic pathways need to be introduced and all requiredenzymes exist in nature. Ethylene oxide will be first converted intoethylene glycol by epoxide hydrolase and this is followed by oxidationto glycoaldehyde. The latter will be converted into acetyl-phosphate.The TPP dependent phosphoketolase will be used to convert glycoaldehydeinto acetyl-phosphate. TPP dependent enzymes are generally promiscuousand known to react with aldehyde intermediates (Demir et al.,Tetrahedron: Asymmetry 10, 4769-4774, 1999). Here, theTPP-glycolaldehyde adduct is an intermediate in the cognate reaction.However, it may be necessary to engineer the active site ofphosphoketolase to enhance its specificity for glycolaldehyde. Thisshould be readily achievable since the reaction is going from a large tosmall substrate, and there are numerous crystal structures available(Suzuki et al., J Biol Chem 285, 34279-34287, 2010). The resultingacetyl-phosphate will be converted into acetyl-coA by phosphateacetyltransferase.

Example 4: Construction of the Ethanol Assimilation Pathway

In order to generate n-butanol from the ethanol produced as describedabove, the synthetic n-butanol pathway described in Atsumi et al., MetabEng 10, 305-311, 2008 and Shen et al., Appl Environ Microbiol 77,2905-2915, 2011 will be combined with pathways 1, 2, and 3 (FIG. 1).

The E. coli strain with this pathway produced more than 30 g/L n-butanolwith 88% theoretical yield. In this pathway, two molecules of acetyl-CoAare condensed into acetoacetyl-CoA by an acetoacetyl-CoA thiolase (AtoB(E. coli)). The acetoacetyl-CoA is reduced to 3-hydroxybutyryl-CoA by anNADH-dependent 3-hydroxybutyryl-CoA dehydrogenase (Hbd (C.acetobutylicum)). A crotonase (Crt (C. acetobutylicum)) then catalyzes adehydration to yield crotonyl-CoA (Waterson et al., J Biol Chem 247,5266-5271, 1972). A trans-enoyl-CoA reductase (Ter (Treponemadenticola)) reduces crotonyl-CoA to butyryl-CoA (Tucci and Martin, FEBSLett 581, 1561-1566, 2007). Lastly, butyryl-CoA is sequentially reducedby a single NADH-dependent aldehyde/alcohol dehydrogenase (AdhE2) ton-butanol (Atsumi et al., Metab Eng 10, 305-311, 2008).

The production of n-butanol from ethanol in the n-butanol-producingstrain described in Shen et al., Appl Environ Microbiol 77, 2905-2915,2011 was attempted. The strain was grown in LB media to an OD₆₀₀ of 0.4,then IPTG (final concentration; 1 mM) was added into the culture toinduce enzymes in n-butanol pathway and incubated at 30° C. After 1 h,the culture was span down and the supernatant was eliminated. The samevolume of M9 media without carbon source as the LB media was added andthe cells were resuspended. Consequently, glucose or ethanol was addedas a solo carbon source into the culture (the final concentration was 10g/L each). These cultures were incubated at 30° C. for n-butanolproduction. OD₆₀₀ and n-butanol concentration of the culture weremeasured at specific time points.

As shown in FIG. 4A, the cell density in not only M9-glucose media butalso M9-ethanol media was found to increase. As AdhE2 in the n-butanolpathway has ability to convert ethanol to acetyl-CoA, the strain couldassimilate ethanol using AdhE2 to grow.

FIG. 4B shows that the strain produced 2.4 mg/L n-butanol in M9-ethanolmedia in 3 days. However, the titer was about 20-times less than inM9-glucose. This result indicated that improving ethanol assimilatingability is necessary to improve n-butanol production from ethanol.

Although E. coli encodes the genes constituting ethanol assimilationpathway, ethanol is not metabolized in wild type E. coli at a sufficientrate to support growth. Certain mutant strains, however, with alteredpattern of gene expression can grow on ethanol as a carbon and energysource. These findings mean that conversion of ethanol to acetyl-CoA isnot efficient in E. coli. To acquire high n-butanol productivity, theconversion efficiency of ethanol to acetyl-CoA has to be improved.Acetyl-CoA is one of the most important metabolite for E. coli to grow.Ethanol conversion efficiency can be estimated by growth of E. coli.

Genes functioning in the ethanol assimilating pathway in E. coli werescreened. Two different ethanol assimilating pathways were constructedusing genes from E. coli and compared. The first pathway consists of twoenzymes: AdhP (ECK1472) and MhpF (ECK0348). AdhP converts ethanol toacetoaldehyde, and then the acetoaldehyde is converted to acetyl-CoA byMhpF. The second pathway is based on only one enzyme: AdhE (ECK1235).AdhE is able to catalyze the reactions catalyzed by both AdhP and MhpF.

To compare these two pathways, strains overexpressing AdhP and MhpF orAdhE were inoculated in M9 minimal media with either glucose or ethanolas the sole carbon source. FIG. 5 shows the growth rate of thesestrains. In M9-glucose media, these strains showed almost same in growthrate (FIG. 5A). On the other hand, growth rates of these strains weresignificantly different in M9-ethanol media (FIG. 5B). The controlstrain without ethanol assimilating pathway was not able to grow inM9-ethanol. The AdhE-overexpressing strain (AdhE strain) andMhpF/AdhP-overexpressing strain (MhpF/AdhP strain) could grow muchbetter than control strain, but the MhpF/AdhP strain grew faster thanthe AdhE strain.

TABLE 3 Screening Genes for Ethanol Assimilation Genes Growth rate inethanol (h⁻¹) mRFP1 0.456 ± 0.014 (in Glucose) adhP 0.026 ± 0.001 mhpF —mhpF-adhP 0.067 ± 0.005 adhE 0.041 ± 0.003 adhE1* — adhE2* 0.054 ± 0.005mhpFE-adhP 0.071 ± 0.001 edgE^(†)-adhP 0.167 ± 0.002 —: no growth, *genefrom Clostridium acetobutylicum, ^(†) Listeria monocytogenes

To further improve the ethanol assimilating pathway, enzymes involved inethanol assimilation were screened (Table 3). Genes or combinations ofgenes were expressed in E. coli, and growth rate of the strains inM9-ethanol was measured. As shown in Table 3, EdgE and AdhP was bestcombination. EdgE is from Listeria monocytogenes and catalyzes reactionfrom acetaldehyde to acetyl-CoA. The growth rate of the strainexpressing EdgE and AdhP with ethanol was about 40% of growth rate withglucose.

Example 5: Production of n-Butanol Using Ethanol Assimilation Pathways

The above results demonstrate that EdgE and AdhP may be used as anefficient ethanol assimilation pathway. EdgE and AdhP were introducedinto an n-butanol-producing strain to produce n-butanol from ethanol.The strains were grown in LB media to an OD₆₀₀ of 0.4, then IPTG (finalconcentration; 1 mM) was added into the culture and incubated at 37° C.After 4 h, the culture was span down and the supernatant was eliminated.The same volume of M9 media without carbon source as the LB media wasadded and the cells were resuspended. Glucose or ethanol was added as asolo carbon source into the culture (the final concentration is 10 g/Leach). These cultures were incubated at 37° C. for 24 h. Consequently,n-butanol concentration in the cultures was measured.

As shown in FIG. 6, In M9-glucose media, an n-butanol-producing strainexpressing EdgE/AdhP (+EdgE/AdhP strain) produced 97 mg/L of n-butanolin 24 h. This titer was about half amount of n-butanol that n-butanolstrain without EdgE/AdhP (−EdgE/AdhP strain) produced (186 mg/L). As+EdgE/AdhP strain produce more ethanol (data not shown), the n-butanolproduction in the strain reduced. In M9-ethanol, on the other hand, 622mg/L or 24 mg/L of n-butanol was produced in +EdgE/AdhP strain or−EdgE/AdhP strain, respectively.

Importantly, the +EdgE/AdhP strain produced about 25-times moren-butanol than the −EdgE/AdhP strain. In addition, the titer ofn-butanol in +EdgE/AdhP strain with ethanol was 3-times higher than in−EdgE/AdhP with glucose. These results indicate that the novelethanol-to-n-butanol pathway described herein is more effective than aglucose-to-n-butanol pathway for the production of n-butanol.

Example 6: Characterization of Ethylene Hydratase Enzymes

As described in Example 1, E. coli optimized synthetic genes for oleatehydratase and 2-haloacrylate hydratase have been cloned into the pET28aexpression vector. These genes have been sequenced to confirm thecorrect sequences, transformed into E. coli, and shown by SDS-PAGE toyield high level expression of the expected enzymes. The specificenzymes investigated are the oleate hydratase (ZP_07049769) fromLysinibacillus fusiformis and the 2-haloacrylate hydratase (BAJ13488)from Pseudomonas sp. As discussed above, oleate hydratase enzymescatalyze the conversion of oleate and water to (R)-10-hydroxystearate,also known as 10-hydroxyoctadecanoic acid (EC 4.2.1.53), whereas2-haloacrylate hydratase enzymes catalyze the conversion of2-chloroacrylate to pyruvate using FADH₂ as a co-factor (FIG. 7).

In order to characterize the activities of these two enzymes inconverting ethylene to ethanol (see “Pathway 1” in FIG. 1), optimizedGC-FID methods were used. For oleate hydratase activity, 50 μg purifiedoleate hydratase was mixed in a reaction with 50 mM PIPES buffer (pH6.5), 0.1 mM FAD, 1 mM Na₂S₂O₄, 5 mM octanoic acid, and water up to 1mL. For 2-haloacrylate hydratase activity, 50 μg purified 2-haloacrylatehydratase was mixed in a reaction with 50 mM PIPES buffer (pH 6.5), 0.1mM FAD, 1 mM Na₂S₂O₄, and water up to 1 mL. After introducing 99%ethylene gas into each reaction for 30 seconds, the reactions wereincubated for 72 hours, then heated to 75° C. for 1 minute. 10 μL of thevial headspace from each reaction was then injected onto the GC-FID.

As shown in FIG. 8, both oleate hydratase and 2-haloacrylate hydrataseproduced ethanol from ethylene, as detected by the presence of ethanolin a vial headspace. No ethanol was detected for either reaction in theabsence of enzyme. These results demonstrate the successful conversionof ethylene to ethanol using oleate hydratase and 2-haloacrylatehydratase.

1. A bacterium comprising a recombinant polynucleotide encoding anethylene hydratase (EH), wherein expression of the EH results in anincrease in production of ethanol as compared to a correspondingbacterium lacking the recombinant polynucleotide.
 2. The bacterium ofclaim 1, wherein the EH is an oleate hydratase.
 3. The bacterium ofclaim 2, wherein the oleate hydratase is a Lysinibacillus fusiformisoleate hydratase.
 4. The bacterium of claim 1, wherein the EH is a2-haloacrylate hydratase.
 5. The bacterium of claim 4, wherein the2-haloacrylate hydratase is a Pseudomonas species 2-haloacrylatehydratase.
 6. The bacterium of claim 1, wherein the EH is a kievitonehydratase.
 7. The bacterium of claim 6, wherein the kievitone hydrataseis a Fusarium solani kievitone hydratase.
 8. A bacterium comprising arecombinant polynucleotide encoding an alkene monooxygenase (AMO) and anethylene oxide reductase (EOR), wherein expression of the AMO and theEOR results in an increase in production of ethanol as compared to acorresponding bacterium lacking the recombinant polynucleotide.
 9. Thebacterium of claim 8, wherein the AMO is a toluene monooxygenase. 10.The bacterium of claim 9, wherein the toluene monooxygenase is aPseudomonas mendocina toluene monooxygenase.
 11. The bacterium of claim9, wherein the toluene monooxygenase is a Burkholderia cepacia toluenemonooxygenase.
 12. The bacterium of claim 8, wherein the EOR is an NAD⁺dependent formate dehydrogenase.
 13. The bacterium of claim 8, whereinthe recombinant polynucleotide comprises a first polynucleotide encodingthe alkene monooxygenase (AMO) and a second polynucleotide encoding theethylene oxide reductase (EOR). 14-21. (canceled)
 22. A bacteriumcomprising a recombinant polynucleotide encoding an alkene monooxygenase(AMO) and an epoxide hydrolase (EPH), wherein expression of the AMO andthe EPH results in an increase in production of ethylene glycol ascompared to a corresponding bacterium lacking the recombinantpolynucleotide.
 23. The bacterium of claim 22, wherein the bacteriumfurther comprises a further recombinant polynucleotide encoding aglycoaldehyde reductase (GR), wherein expression of the AMO, the EPH,and the GR results in an increase in production of glycoaldehyde ascompared to a corresponding bacterium lacking the recombinantpolynucleotides.
 24. The bacterium of claim 23, wherein the bacteriumfurther comprises a still further recombinant polynucleotide encoding aphosphoketolase (PK), wherein expression of the AMO, the EPH, the GR,and the PK results in an increase in production of acetyl-phosphate ascompared to a corresponding bacterium lacking the recombinantpolynucleotides.
 25. The bacterium of claim 24, wherein the bacteriumfurther comprises a yet further recombinant polynucleotide encoding aphosphate acetyltransferase (PA), wherein expression of the AMO, theEPH, the GR, the PK, and the PA results in an increase in production ofacetyl-CoA as compared to a corresponding bacterium lacking therecombinant polynucleotides.
 26. The bacterium of claim 25, wherein thebacterium further comprises a yet still further recombinantpolynucleotides encoding an acetoacetyl-CoA thiolase (AT), a3-hydroxybutyryl-CoA dehydrogenase (HBD), a crotonase (CRT), atrans-enoyl-CoA reductase (TER), and an alcohol/aldehyde dehydrogenase(AADH), wherein expression of the AT, the HBD, the CRT, the TER, and theAADH results in an increase in production of n-butanol as compared to acorresponding bacterium lacking the recombinant polynucleotides. 27-33.(canceled)
 34. A method for producing ethanol, the method comprising: a)providing the bacterium of claim 1; and b) culturing the bacterium of(a) in culture medium comprising a substrate under conditions suitablefor the conversion of the substrate to ethanol, wherein expression ofthe EH results in an increase in production of ethanol as compared to acorresponding bacterium lacking the recombinant polynucleotide, whencultured under the same conditions.
 35. The method of claim 34, furthercomprising step (c) substantially purifying the ethanol.
 36. A methodfor producing n-butanol, the method comprising: a) providing thebacterium of claim 26; and b) culturing the bacterium of (a) in culturemedium comprising a substrate under conditions suitable for theconversion of the substrate to n-butanol, wherein expression of theenzymes encoded by the recombinant polynucleotides results in anincrease in production of n-butanol as compared to a correspondingbacterium lacking the recombinant polynucleotides, when cultured underthe same conditions.
 37. The method of claim 36, further comprising step(c) substantially purifying the n-butanol. 38-40. (canceled)
 41. Amethod for producing ethanol, the method comprising: a) providing thebacterium of claim 6; and b) culturing the bacterium of (a) in culturemedium comprising a substrate under conditions suitable for theconversion of the substrate to ethanol, wherein expression of the AMOand the EOR results in an increase in production of ethanol as comparedto a corresponding bacterium lacking the recombinant polynucleotide,when cultured under the same conditions.
 42. The method of claim 41,further comprising step (c) substantially purifying the ethanol.